Methods of treating myeloproliferative disorders

ABSTRACT

The present disclosure relates to the use of 2-((4S)-6-(4-chlorophenyl)-1-methyl-4H-benzo[c]isoxazolo[4,5-e]azepin-4-yl)acetamide, and pharmaceutically acceptable salts thereof, for treating myelofibrosis.

RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/US2019/063515, filed Nov. 27, 2019, which claims priority toInternational Application No. PCT/US2018/062534, filed Nov. 27, 2018 andInternational Application No. PCT/US2019/059784, filed Nov. 5, 2019, theentire contents of each of which are incorporated herein by reference.

BACKGROUND

Myeloproliferative disorders are diseases of the bone marrow and blood.Myelofibrosis, for example, is a clonal myeloproliferative disease thatis characterized by exaggerated abnormalities in megakaryocytes. Theabnormal megakaryocytes are attributed primarily to dysregulation of theJAK/STAT pathway, although there is dysregulation in a number of otherpathways as well. Due to the multiple pathways affected and the array ofdownstream effects, myelofibrosis is a complex, heterogeneous diseasewith many inter-related features. The abnormal megakaryocytes releaseexcess platelets and cytokines, both pro-inflammatory and pro-fibrotic(transforming growth factor beta [TGF-β]), into the bone marrow. Thepro-inflammatory cytokines lead to debilitating constitutional symptomsand exacerbate the deposition of collagen signaled by pro-fibroticpathways. Bone marrow fibrosis is the hallmark of myelofibrosis,although diagnosis is not necessarily dependent on it. The bone marrowfibrosis is the key feature that causes the morbidity and mortalityassociated with the disease. The bone marrow fibrosis and inflammatorystate of myelofibrosis often lead to cytopenias, extramedullaryhematopoiesis (EMH), organomegaly such as splenomegaly and hepatomegalyand a myriad of constitutional symptoms.

Myelofibrosis is a serious disease in that it is both life-threateningand greatly diminishes the quality of life of the patient before itaffects survival. The two most common causes of death are conversion toacute myeloid leukemia (AML) and progression of the disease. Thetreatment paradigm is dictated by the number of risk factors present,which then correlate with different survival rates. While allogeneichematopoietic stem cell transplantation (HCT) can be curative, it isassociated with its own morbidity and mortality, which limit its use tothose eligible patients whose prognosis is worse (<5 years) than therisk of moving forward with the transplant. The remaining treatments aremore palliative in nature, either due to their mechanism of action(e.g., treatments specifically focused on the anemia that is frequentlyassociated with myelofibrosis) or due to the restricted effects that thetreatment can elicit (e.g., the standard of care ruxolitinib).

Ruxolitinib, a JAK1/2 inhibitor, is approved for the treatment ofmyelofibrosis. JAK is a key regulator in hematopoiesis, immuneregulation, growth and embryogenesis (Stahl M, Zeidan AM (2017).Management of Myelofibrosis: JAK Inhibition and Beyond. Expert RevHematol; 17(5): 459-477). Dysregulated JAK signaling can lead toincreased thrombopoietin signaling, which is believed to be one of thecauses of increased megakaryocyte production and platelets inmyelofibrosis. Further, JAK signaling is implicated in the release ofpro-inflammatory cytokines and growth factors that cause constitutionalsymptoms and splenomegaly: JAK-1 plays a role in the signaling ofpro-inflammatory cytokines (e.g., IL-1, IL-16, TNF-α), the cause ofsystemic symptoms in myelofibrosis and JAK-2 impacts growth factors andother cytokines (e.g., IL-3, IL-5) that are believed to promotesplenomegaly in myelofibrosis. Through this mechanism of action,ruxolitinib has demonstrated an ability to reduce the spleen volumes andsymptoms of myelofibrosis patients, thereby improving their quality oflife. Unfortunately, however, there are a number of limitations with thecurrent use of ruxolitinib.

Ruxolitinib is considered a palliative treatment due to its lack ofdisease-modifying effects. It does not affect the mutant allele burdenor bone marrow fibrosis (Novel Therapies for Myelofibrosis, 2017, CurrHematol Malig Rep; 12(6): 611-624). In addition, constitutional symptomswill revert back after a week off of ruxolitinib treatment (see TefferiA (2017); Management of Primary Myelofibrosis; UpToDate; 1-23). Next,anemia negatively impacts patient quality of life, has the highest powerof predicting shortened survival, and limits access to optimal standardof care. Ruxolitinib is not a viable treatment option for some anemicpatients because ruxolitinib is known to decrease red blood cellproduction and hemoglobin levels. Anemic patients, for example, areeither not treated at all with ruxolitinib, given a lower dose ofruxolitinib leading to inadequate response, or give a full doseruxolitinib, which typically leads to need for red blood cell (RBC)transfusions. See e.g., Haematologica. 2016 December; 101(12):e482-e484. Patients who have become dependent on RBC transfusions sufferfrom an even worse quality of life and prognosis.

Another unmet medical need is the lack of alternative therapies fortreating myelofibrosis. This means that i) those who do not achieve anadequate response to ruxolitinib; ii) those who are intolerant toruxolitinib; and iii) those who progress despite treatment withruxolitinib have little or no alternative treatment options.Furthermore, approximately 75% of patients who do initially respond toruxolitinib end up discontinuing treatment due to disease progression ortoxicity.

SUMMARY

It has now been found that2-((4S)-6-(4-chlorophenyl)-1-methyl-4H-benzo[c]isoxazolo[4,5-e]azepin-4-yl)acetamide,an inhibitor of the Bromodomain and Extra-Terminal (BET) family andreferenced herein as Compound 1, is effective in treating myelofibrosisand has numerous advantages over the current standard of care, i.e.,ruxolitinib.

Unlike ruxolitinib, treating myelofibrosic subjects with Compound 1increased hemoglobin levels. This is particularly important for subjectswho are also anemic. For example, patients 247 and 248 in theExemplification section below experienced an increase in hemoglobinlevels from about 8 g/dL to near normal at about 11.5 g/dL. See e.g.,FIG. 5. In addition, platelet counts were normalized from about 8 g/dlto about 10.9 g/dl. See e.g., FIG. 5.

Other results showed that uncontrolled thrombocytosis could bealleviated (i.e., platelets were normalized) in a subject that wasrefractory to all standard of care, including the JAK inhibitorruxolitinib, following treatment with Compound 1. See e.g., FIG. 6 andthe Exemplification section below. An improvement in headaches was alsofound.

Further results showed transfusion dependence could be reversedfollowing treatment with Compound 1. For example, the subject who wastransfusion dependent while taking the JAK inhibitor ruxolitinib becametransfusion independent after undergoing treatment with Compound 1, andremained transfusion independent for more than 24 weeks. See e.g., FIG.5. Similar results were seen using a combination of both ruxolitinib andCompound 1. See e.g., Patients 245 and 246 in the Exemplificationsection below and FIG. 6, as well as the expanded data provided e.g., inFIG. 8, FIG. 10A, and FIG. 10B.

As an additional advantage, Compound 1 significantly decreased spleensize, even in subjects who were resistant to ruxolitinib. For example,prior to administration of Compound 1, Patient 245 as described belowbecame resistant to ruxolitinib with her spleen increasing 25% in size(spleen volume was 12 cm by palpation). However, after 4 weeks oftherapy with Compound 1 and ruxolitinib, her spleen size was reduced to5 cm. See also FIG. 8, FIG. 9 FIG. 10A, and FIG. 12A which shows thespleen reduction from a Phase 2 human trial with Compound 1 andruxolitinib.

Provided herein therefore are methods of using Compound 1, or apharmaceutically acceptable salt thereof, alone or in combination with aJAK inhibitor such as ruxolitinib, to treat myelofibrosis.

In certain aspects, also provided herein are methods of using Compound1, or a pharmaceutically acceptable salt thereof, alone or incombination with a JAK inhibitor such as ruxolitinib, to treatmyelofibrosis in subjects with anemia.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the effects of Compound 1 on IL6 and IL10 mRNA transcriptlevels.

FIG. 2 depicts histograms of Compound 1 effect on megakaryocytedifferentiation.

FIG. 3 represents the histograms and quantitation of effects on maturemegakaryocyte marker CD42b after Treatment with Compound 1 andruxolitinib for 10 days in stem-cell derived megakaryocyte cultures fromhealthy donor 2 where the grey histrogram is DMSO treated sample, bluehistogram is Compound 1 treated sample, and CD42b high calculationsrefer to the Compound 1-treated samples.

FIG. 4 shows the repression of BET-target genes IL8 and CCR1 incirculating blood 2 hours post-dose as a function of the plasmaconcentration of Compound 1.

FIG. 5 shows the changes in hemoglobin levels and transfusionrequirements in a combination arm of Compound 1 and ruxolitinib.

FIG. 6 shows the change in platelet and hemoglobin levels in patient 247of the monotherapy arm.

FIG. 7 shows the study design for a Phase 2 trial with Compound 1 andCompound 1 with ruxolitinib in patients with myelofibrosis.

FIG. 8 shows the spleen reduction volume improvement from a Phase 2human trial using Compound 1 monotherapy or as an add-on to ruxolitinibin myelofibrosis patients (panel A) as well as the total symptom score(TSS) improvement from a Phase 2 human trial using Compound 1monotherapy or as an add-on to ruxolitinib in myelofibrosis patients(panel B).

FIG. 9 shows the spleen reduction volume and total symptom score (TSS)improvement from a Phase 2 human trial using Compound 1 and ruxolitinibin JAK inhibitor treatment naïve myelofibrosis patients.

FIG. 10A illustrates the percent spleen reduction volume at 24 weeksafter treatment with Compound 1 as an add-on to ruxolitinib (Arm 2) inpatients with refractory or intolerant myelofibrosis subjects who weretransfusion dependent at the start of therapy.

FIG. 10B illustrates the percent total symptom score improvement aftertreatment with Compound 1 as an add-on to ruxolitinib (Arm 2) inpatients with refractory or intolerant myelofibrosis subjects who weretransfusion dependent at the start of therapy.

FIG. 10C illustrates the Patient Global Impression of Change aftertreatment with Compound 1 as an add-on to ruxolitinib (Arm 2) inpatients with refractory or intolerant myelofibrosis subjects who weretransfusion dependent at the start of therapy.

FIG. 11A illustrates the absolute hemoglobin and transfusion requirementfor patient 11-246 following treatment with Compound 1 as an add-on toruxolitinib (Arm 2).

FIG. 11B illustrates the absolute hemoglobin and transfusion requirementfor patient 19-277 following treatment with Compound 1 as an add-on toruxolitinib (Arm 2).

FIG. 11C illustrates the absolute hemoglobin and transfusion requirementfor patient 11-252 following treatment with Compound 1 as an add-on toruxolitinib (Arm 2).

FIG. 11D illustrates the absolute hemoglobin and transfusion requirementfor patient 12-294 following treatment with Compound 1 as an add-on toruxolitinib (Arm 2).

FIG. 12A illustrates the percent spleen reduction volume at 24 weeksafter treatment with Compound 1 as an add-on to ruxolitinib (Arm 2) inpatients with refractory or intolerant myelofibrosis who werenon-transfusion dependent at the start of therapy.

FIG. 12B illustrates the percent total symptom score improvement aftertreatment with Compound 1 as an add-on to ruxolitinib (Arm 2) inpatients with refractory or intolerant myelofibrosis who werenon-transfusion dependent at the start of therapy.

FIG. 12C illustrates the Patient Global Impression of Change aftertreatment with Compound 1 as an add-on to ruxolitinib (Arm 2) inpatients with refractory or intolerant myelofibrosis who werenon-transfusion dependent at the start of therapy.

FIG. 13A illustrates the percent spleen reduction volume at 24 weeksafter treatment with Compound 1 monotherapy (Arm 1) in patients withrefractory or intolerant myelofibrosis who were transfusion (Cohort 1A)or non-transfusion dependent (Cohort 1B) at the start of therapy.

FIG. 13B illustrates the percent total symptom score improvement aftertreatment with Compound 1 monotherapy (Arm 1) in patients withrefractory or intolerant myelofibrosis who were transfusion (Cohort 1A)or non-transfusion dependent (Cohort 1B) at the start of therapy.

FIG. 13C illustrates the Patient Global Impression of Change aftertreatment with Compound 1 monotherapy (Arm 1) in patients withrefractory or intolerant myelofibrosis who were transfusion (Cohort 1A)or non-transfusion dependent (Cohort 1B) at the start of therapy.

FIG. 14 shows the improvement in bone marrow fibrosis from Arms 1 and 2of the phase 2 clinical trial using Compound 1 monotherapy or as anadd-on to ruxolitinib in patients with refractory or intolerantmyelofibrosis who were transfusion or non-transfusion dependent at thestart of therapy.

FIG. 15A illustrates the reduction of CRP in patients with refractory orintolerant myelofibrosis who were transfusion or non-transfusiondependent at the start of therapy and were treated with Compound 1alone, i.e., monotherapy.

FIG. 15B illustrates the reduction of IL-8 in patients with refractoryor intolerant myelofibrosis who were transfusion or non-transfusiondependent at the start of therapy and were treated with Compound 1alone, i.e., monotherapy.

FIG. 15C illustrates the reduction of IL-18 in patients with refractoryor intolerant myelofibrosis who were transfusion or non-transfusiondependent at the start of therapy and were treated with Compound 1alone, i.e., monotherapy.

FIG. 15D illustrates the reduction of CRP in patients with refractory orintolerant myelofibrosis who were transfusion or non-transfusiondependent at the start of therapy and were treated with Compound 1 as anadd-on to ruxolitinib.

FIG. 15E illustrates the reduction of IL-8 in patients with refractoryor intolerant myelofibrosis who were transfusion or non-transfusiondependent at the start of therapy and were treated with Compound 1 as anadd-on to ruxolitinib.

FIG. 15F illustrates the reduction of IL-18 in patients with refractoryor intolerant myelofibrosis who were transfusion or non-transfusiondependent at the start of therapy and were treated with Compound 1 as anadd-on to ruxolitinib.

FIG. 16 illustrates the percent spleen reduction volume at 12 weeksafter treatment with Compound 1 in JAK inhibitor naïve (Arm 3) patientswith myelofibrosis.

FIG. 17 illustrates the percent total symptom score improvement aftertreatment with Compound 1 in JAK inhibitor naïve (Arm 3) patients withmyelofibrosis.

FIG. 18 illustrates the Patient Global Impression of Change aftertreatment with Compound 1 in JAK inhibitor naïve (Arm 3) patients withmyelofibrosis.

DETAILED DESCRIPTION

2-((4S)-6-(4-chlorophenyl)-1-methyl-4H-benzo[c]isoxazolo[4,5-e]azepin-4-yl)acetamideis exemplified as Compound 144 in U.S. Pat. No. 8,796,261, the entirecontents of which are incorporated herein by reference.2-((4S)-6-(4-chlorophenyl)-1-methyl-4H-benzo[c]isoxazolo[4,5-e]azepin-4-yl)acetamideis used interchangeably herein with Compound 1 and/or CPI-0610, and isrepresented by the following structural formula:

Crystalline forms of Compound 1 are disclosed in U.S. Pat. No.9,969,747, the entire contents of which are incorporated by referenceherein.

Compound 1 is a potent and selective small molecule designed to promoteanti-tumor activity by selectively inhibiting the function of BETprotein. See e.g., J. Med. Chem., 2016; Feb. 25; 59(4): 1330-9. Compound1 is being investigated for its profound effects in treatinghematological malignancies including progressive lymphoma. See e.g.,U.S. Clinical Trials NCT02157636 and NCT01949883. It has now been found,however, that Compound 1 is also effective in treating myelofibrosis. Tothis end, for example, Compound 1 increased hemoglobin levels,normalized platelet counts, and reduced spleen size. Subjects who werepreviously transfusion dependent became transfusion independent aftertreatment.

Therefore, in a first embodiment, provided herein is a method oftreating myelofibrosis in a subject comprising administering to thesubject a therapeutically effective amount of2-((4S)-6-(4-chlorophenyl)-1-methyl-4H-benzo[c]isoxazolo[4,5-e]azepin-4-yl)acetamide,or a pharmaceutically acceptable salt thereof. Also provided is the useof2-((4S)-6-(4-chlorophenyl)-1-methyl-4H-benzo[c]isoxazolo[4,5-e]azepin-4-yl)acetamide,or a pharmaceutically acceptable salt thereof, in the manufacture of amedicament for treating myelofibrosis in a subject. Further provided is2-((4S)-6-(4-chlorophenyl)-1-methyl-4H-benzo[c]isoxazolo[4,5-e]azepin-4-yl)acetamide,or a pharmaceutically acceptable salt thereof, for treatingmyelofibrosis in a subject.

The terms “subject” and “patient” may be used interchangeably, and meana mammal in need of treatment, e.g., companion animals (e.g., dogs,cats, and the like), farm animals (e.g., cows, pigs, horses, sheep,goats and the like) and laboratory animals (e.g., rats, mice, guineapigs and the like). Typically, the subject is a human in need oftreatment.

The terms “treatment,” “treat,” and “treating” refer to reversing,alleviating, reducing the likelihood of developing, or inhibiting theprogress of myelofibrosis, or one or more symptoms thereof, as describedherein. In some embodiments, treatment may be administered after one ormore symptoms have developed, i.e., therapeutic treatment. In otherembodiments, treatment may be administered in the absence of symptoms.For example, treatment may be administered to a susceptible individualprior to the onset of symptoms (e.g., in light of a history of symptomsand/or in light of genetic or other susceptibility factors), i.e.,prophylactic treatment. Treatment may also be continued after symptomshave resolved, for example to prevent or delay their recurrence.Symptoms specific to myelofibrosis include, but are not limited to,abdominal discomfort, dyspnea on exertion, early satiety, fatigue,headaches, night sweats, dizziness, fever, chills, insomnia, pruritus,or bone pain.

As detailed in the Exemplification section below, Compound 1 waseffective in subjects who have undergone treatment for myelofibrosiswith JAK inhibitors such as ruxolitinib. Therefore, in a secondembodiment, provided herein is a method of treating myelofibrosis in asubject comprising administering to the subject a therapeuticallyeffective amount of2-((4S)-6-(4-chlorophenyl)-1-methyl-4H-benzo[c]isoxazolo[4,5-e]azepin-4-yl)acetamide,or a pharmaceutically acceptable salt thereof, wherein the subject haspreviously undergone treatment with a janus kinase (JAK) inhibitor(e.g., ruxolitinib). Also provided is the use of2-((4S)-6-(4-chlorophenyl)-1-methyl-4H-benzo[c]isoxazolo[4,5-e]azepin-4-yl)acetamide,or a pharmaceutically acceptable salt thereof, in the manufacture of amedicament for treating myelofibrosis in a subject who has previouslyundergone treatment with janus kinase (JAK) inhibitor (e.g.,ruxolitinib). Further provided is2-((4S)-6-(4-chlorophenyl)-1-methyl-4H-benzo[c]isoxazolo[4,5-e]azepin-4-yl)acetamide,or a pharmaceutically acceptable salt thereof, for treatingmyelofibrosis in a subject who has previously undergone treatment with ajanus kinase (JAK) inhibitor (e.g., ruxolitinib).

As detailed in the Exemplification section below, Compound 1 waseffective in subjects who have myelofibrosis, but are JAK inhibitorsnaïve subjects. Therefore, in a third embodiment, provided herein is amethod of treating myelofibrosis in a subject comprising administeringto the subject a therapeutically effective amount of2-(((4S)-6-(4-chlorophenyl)-1-methyl-4H-benzo[c]isoxazolo[4,5-e]azepin-4-yl)acetamide,or a pharmaceutically acceptable salt thereof, wherein the subject isjanus kinase (JAK) inhibitor naïve subject. Also provided is the use of2-((4S)-6-(4-chlorophenyl)-1-methyl-4H-benzo[c]isoxazolo[4,5-e]azepin-4-yl)acetamide,or a pharmaceutically acceptable salt thereof, in the manufacture of amedicament for treating myelofibrosis in a subject who is a janus kinase(JAK) inhibitor naïve subject. Further provided is2-((4S)-6-(4-chlorophenyl)-1-methyl-4H-benzo[c]isoxazolo[4,5-e]azepin-4-yl)acetamide,or a pharmaceutically acceptable salt thereof, for treatingmyelofibrosis in a subject who is a janus kinase (JAK) inhibitor naïvesubject.

In a fourth embodiment, the subjects described in the first and secondembodiments are characterized as progressed/relapsed to a JAK inhibitor.In a fifth embodiment, the subjects described in the first and secondembodiments are characterized is refractory/resistant to a JAKinhibitor. Alternatively, as part of a fifth embodiment, the subjectsdescribed in the first and second embodiments are characterized asintolerant to a JAK inhibitor.

A subject who is characterized as progressed/relapsed is one who at onetime responded to treatment with a JAK inhibitor (e.g., ruxolitinib),but who no longer responds. A subject who is characterized asrefractory/resistant is one who is unresponsive or demonstratesworsening of disease while on treatment with a JAK inhibitor (e.g.,ruxolitinib). In one aspect, evidence of refractoriness/resistance(including loss of response) includes no spleen size reduction orsymptom improvement after 6 months of therapy, disease progression, orintolerance to ruxolitinib (i.e., platelet count <50×10⁹/L and/or ANC<0.5×10⁹/L despite recommended dose adjustments and interruptions perapproved ruxolitinib label; bleeding; or other severe [i.e. ≥Grade 3non-hematological] toxicity).

A subject who is characterized as intolerant is one who cannot toleratethe side effects from treatment with a JAK inhibitor (e.g., ruxolitinib)and thus has to be removed from treatment of said JAK inhibitor.

An illegible patient is defined as those patients for whom a JAKinhibitor is indicated (e.g., ruxolitinib), but the healthcare provideris reluctant to initiate treatment with the JAK inhibitor due to priorhistory of severe infections such as tuberculosis, PML, or skinmalignancies that are known to be associated or exacerbated by the JAKinhibitor (see e.g., the approved package insert for ruxolitinib).

Compound 1 was also shown to be effective as a combination treatmentwith the JAK inhibitor ruxolitinib. Therefore, in a sixth embodiment,provided herein is a method of treating myelofibrosis in a subjectcomprising administering to the subject a therapeutically effectiveamount of2-((4S)-6-(4-chlorophenyl)-1-methyl-4H-benzo[c]isoxazolo[4,5-e]azepin-4-yl)acetamideand a therapeutically effective amount of a janus kinase (JAK) inhibitor(e.g., ruxolitinib), or a pharmaceutically acceptable salt of any of theforegoing. Also provided is the use of2-((4S)-6-(4-chlorophenyl)-1-methyl-4H-benzo[c]isoxazolo[4,5-e]azepin-4-yl)acetamideand a janus kinase (JAK) inhibitor (e.g., ruxolitinib), or apharmaceutically acceptable salt of any of the foregoing, in themanufacture of a medicament for treating myelofibrosis in a subject.Further provided is2-((4S)-6-(4-chlorophenyl)-1-methyl-4H-benzo[c]isoxazolo[4,5-e]azepin-4-yl)acetamideand a janus kinase (JAK) inhibitor (e.g., ruxolitinib), or apharmaceutically acceptable salt of any of the foregoing, for treatingmyelofibrosis in a subject. Alternatively, as part of a sixthembodiment, the subject to be treated is a janus kinase (JAK) inhibitornaïve subject prior to treatment.

As used herein, ruxolitinib refers to the JAK inhibitor(R)-3-(4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrilephosphate having the following formula.

The term “effective amount” or “therapeutically effective amount” areused interchangeably and include an amount of a compound describedherein that will elicit a desired medical response in a subject havingmyelofibrosis, e.g., reducing the symptoms of and/or slowing theprogression of the disease.

In a seventh embodiment, the subject treated by the methods describedherein (e.g., as in any one of the first through sixth embodiments) iscytopenic. Cytopenic refers to subjects in which the production of oneor more blood cell types ceases or is greatly reduced. Types ofcytopenia include e.g., anemia (a deficiency of red blood cells),leukopenia or neutropenia (a deficiency of white blood cells),thrombocytopenia (a deficiency in the platelets), and pancytopenia (adeficiency in all three of red blood cells, white blood cells, andplatelet counts).

In an eighth embodiment, the subject treated by the methods describedherein (e.g., as in any one of the first through seventh embodiments) isanemic. A subject of the present disclosure (e.g., as in any one of thefirst through seventh embodiments) is said to be anemic if theirhemoglobin value is less than 13.5 g/dL of blood for a male subject orless than 12.0 g/dL of blood for a female subject. In some aspects, asubject (e.g., as in any one of the first through seventh embodiments)is defined herein as being anemic if their hemoglobin value is less than10.0 g/dL. Subjects treatable by the present methods (e.g., as in anyone of the first through seventh embodiments) therefore include thosehaving hemoglobin values less than 13.0 g/dL, less than 12.5 g/dL, lessthan 12.0 g/dL, less than 11.5 g/dL, less than 11.0 g/dL, less than 10.5g/dL, less than 10.0 g/dL, less than 9.5 g/dL, less than 9.0 g/dL, orless than 8.5 g/dL for male subjects and less than 11.5 g/dL, less than11.0 g/dL, less than 10.5 g/dL, less than 10.0 g/dL, less than 9.5 g/dL,less than 9.0 g/dL, or less than 8.5 g/dL for female subjects. In othersaspects, a subject (e.g., as in any one of the first through seventhembodiments) is defined herein as being anemic if their hemoglobin valueranges from 7.5 g/dL of blood to 13.5 g/dL of blood for a male subjector from 7.5 g/dL of blood to 12.0 g/dL of blood for a female subject. Inothers aspects, a subject (e.g., as in any one of the first throughseventh embodiments) is defined herein as being anemic if theirhemoglobin value ranges from 7.5 g/dL of blood to 10.5 g/dL of blood fora male subject or from 7.5 g/dL of blood to 10.5 g/dL of blood for afemale subject. In others aspects, a subject (e.g., as in any one of thefirst through seventh embodiments) is defined herein as being anemic iftheir hemoglobin value ranges from 7.5 g/dL of blood to 10.0 g/dL ofblood for a male subject or from 7.5 g/dL of blood to 10.0 g/dL of bloodfor a female subject. In others aspects, a subject (e.g., as in any oneof the first through seventh embodiments) is defined herein as beinganemic if their hemoglobin value ranges from 7.7 g/dL of blood to 10.7g/dL of blood for a male subject or from 7.7 g/dL of blood to 10.5 g/dLof blood for a female subject. In others aspects, a subject (e.g., as inany one of the first through seventh embodiments) is defined herein asbeing anemic if their hemoglobin value ranges from 7.7 g/dL of blood to10.0 g/dL of blood for a male subject or from 7.7 g/dL of blood to 10.0g/dL of blood for a female subject.

In a ninth embodiment, subjects treated by the methods described herein(e.g., as in any one of the first through eighth embodiments) arethrombocytopenic. A subject of the present disclosure (e.g., as in anyone of the first through eighth embodiments) is said to bethrombocytopenic if their platelet count is less than 150,000platelets/4, of blood. Subjects treatable by the present methods (e.g.,as in any one of the first through eighth embodiments) therefore includethose having platelet levels less than 140,000 platelets/μL, less than130,000 platelets/μL, less than 120,000 platelets/μL, less than 110,000platelets/μL, less than 100,000 platelets/μL, less than 90,000platelets/μL, less than 80,000 platelets/μL, less than 70,000platelets/μL, less than 60,000 platelets/μL or less than 50,000platelets/μL, alone or in combination with one or more of the hemoglobinvalues described above.

In a tenth embodiment, subjects treated by the methods described herein(e.g., as in any one of the first through eighth embodiments) arethrombocytemic. A subject of the present disclosure treated by themethods described herein (e.g., as in any one of the first througheighth embodiments) is said to be thrombocytemic if their platelet countis more than 450,000 platelets/μL of blood. Subjects treatable by thepresent methods (e.g., as in any one of the first through eighthembodiments) therefore include those having platelet levels more than450,000 platelets/μL, more than 500,000 platelets/μL, more than 550,000platelets/μL, or more than 600,000 platelets/μL, alone or in combinationwith one or more of the hemoglobin values described above.Alternatively, as part of a tenth embodiment, a subject of the presentdisclosure treated by the methods described herein (e.g., as in any oneof the first through eighth embodiments) is said to be thrombocytemic iftheir platelet count is more than 400,000 platelets/μL of blood.Subjects treatable by the present methods (e.g., as in any one of thefirst through eighth embodiments) therefore include those havingplatelet levels more than 400,000 platelets/μL.

In an eleventh embodiment, the subject treated by the methods describedherein (e.g., as in any one of the first through tenth embodiments) isleukopenic. A subject (e.g., as in any one of the first through tenthembodiments) is said to be leukopenic if their white blood cell (WBC)count is less than 4,000 WBCs/μ1_, of blood. In certain aspects,subjects treatable by the present methods (e.g., as in any one of thefirst through tenth embodiments) include those having WBC counts of lessthan 3,500 WBCs/μL, 3,200 WBCs/μL, 3,000 WBCs/μL, or 2,500 WBCs/μL,alone or in combination with one or more of the hemoglobin and/orplatelet values described above.

In a twelfth embodiment, the subject treated by the methods describedherein is (e.g., as in any one of the first through eleventhembodiments) neutropenic. In one aspect, a subject of the presentdisclosure (e.g., as in any one of the first through eleventhembodiments) is said to be neutropenic if their neutrophil count is lessthan 1500 neutrophils/μ1_, of blood. In certain aspects, subjectstreatable by the present methods (e.g., as in any one of the firstthrough eleventh embodiments) include those having neutrophil counts ofless than 1250 neutrophils/μL, 1000 neutrophils/μL, 750 neutrophils/μL,or 500 neutrophils/μL, alone or in combination with one or more of thehemoglobin, platelet, and/or WBC values described above.

Myelofibrosis is often associated with an enlarging of the spleen.Enlarging of the spleen can result in a feeling of fullness,indigestion, and a loss of appetite. In a thirteenth embodiment,subjects treatable by the present methods (e.g., as in any one of thefirst through twelfth embodiments) include those having an enlargedspleen or liver.

In a fourteenth embodiment, subjects treatable by the present methods(e.g., as in any one of the first through thirteenth embodiments) mayalso be experiencing one or more additional symptoms. These symptomsinclude, but are not limited to, abdominal discomfort, dyspnea onexertion, early satiety, fatigue, headaches, night sweats, dizziness,insomnia, pruritus, or bone pain.

In a fifteenth embodiment, subjects treated by the present methods(e.g., as in any one of the first through fourteenth embodiments) aretransfusion dependent prior to treatment with Compound 1. In someaspects, “transfusion dependent” means that a subject requires red bloodcell (RBC) transfusions in order to maintain an acceptable level ofhemoglobin. An acceptable level of hemoglobin is determined by thoseskill in the art and can range from e.g., from 13.5 to 17.5 g/dL ofblood for men and from 12.0 to 15.5 g/dL of blood in women. It will beunderstood that subjects undergoing treatment with Tux may have lowerhemoglobin levels than those described above and still be deemed an“acceptable” level in order for treatment to continue.

In a sixteenth embodiment, subjects treated by the present methods(e.g., as in any one of the first through fifteenth embodiments)experience a reduction in spleen size. In one aspect, the reductioncomprises a 10% or more (e.g., a 15% or more, a 20% or more, a 25% ormore, a 30% or more, a 35% or more, a 40% or more, a 45% or more, a 50%or more, a 55% or more, a 60% or more, or a 65% or more reduction inspleen volume from baseline. In another aspect, the reduction comprisesfrom a 10% to a 65% reduction in spleen volume from baseline.

In a seventeenth embodiment, subjects treated by the present methods(e.g., as in any one of the first through sixteenth embodiments)experience a reduction in headaches.

In an eighteenth embodiment, subjects treated by the present methods(e.g., as in any one of the first through seventeenth embodiments) havea reduction in the number of blood transfusion.

In a nineteenth embodiment, subjects treated by the present methods(e.g., as in any one of the first through eighteenth embodiments)experience a normalization of platelets.

In a twentieth embodiment, subjects treated by the present methods(e.g., as in any one of the first through nineteenth embodiments)experience an increase in hemoglobin values.

In a twenty-first embodiment, subjects treated by the present methods(e.g., as in any one of the first through twentieth embodiments)experience an improvement in bone marrow fibrosis as determined e.g., bythe bone marrow fibrosis grading scale (see Thiele J et al.,Haematologica, 2005, 90, 1128). In one aspect, an improvement is definedas at least one grade improvement in the bone marrow fibrosis/reticulingrading compared to baseline.

In a twenty-second embodiment, subjects treated by the present methods(e.g., as in any one of the first through twenty-second embodiments)experience a reduction in pro-inflammatory cytokines such as e.g., CRP,IL-8, and/or IL-18.

The compounds of the methods described herein can be formulated aspharmaceutical compositions and administered to a subject, such as ahuman, in a variety of forms adapted to the chosen route ofadministration. Typical routes of administering such pharmaceuticalcompositions include, without limitation, oral, topical, buccal,transdermal, inhalation, parenteral, sublingual, rectal, vaginal, andintranasal. The term parenteral as used herein includes subcutaneousinjections, intravenous, intramuscular, intrathecal, intrasternalinjection or infusion techniques. Methods of formulating pharmaceuticalcompositions are well known in the art, for example, as disclosed in“Remington: The Science and Practice of Pharmacy,” University of theSciences in Philadelphia, ed., 21st edition, 2005, Lippincott, Williams& Wilkins, Philadelphia, Pa.

Pharmaceutical compositions of the invention can be prepared bycombining a compound of the methods described herein with an appropriatepharmaceutically acceptable carrier, diluent or excipient, and may beformulated into preparations in solid, semi-solid, liquid or gaseousforms, such as tablets, capsules, powders, granules, ointments,solutions, suppositories, injections, inhalants, gels, microspheres, andaerosols. Thus, the present compounds of the methods described hereinmay be systemically administered, e.g., orally, in combination with apharmaceutically acceptable excipient such as an inert diluent or anassimilable edible carrier. They may be enclosed in hard or soft shellgelatin capsules, may be compressed into tablets or may be incorporateddirectly with the food of the patient's diet. For oral therapeuticadministration, the active compound may be combined with one or moreexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.

A specific dosage and treatment regimen for any particular patient willdepend upon a variety of factors, including the activity of the specificcompound employed, the age, body weight, general health, sex, diet, timeof administration, rate of excretion, drug combination, and the judgmentof the treating physician and the severity of the particular diseasebeing treated. The amount of a compound described herein in thecomposition will also depend upon the particular compound in thecomposition. In one aspect, however, when used as a monotherapy (i.e.,without a JAK inhibitor such as ruxolitinib) Compound 1, or apharmaceutically acceptable salt thereof, may be formulated at a dose offrom 50 mg to 500 mg for e.g., administration once, twice, or threetimes daily. For example, in monotherapies, Compound 1 may beadministered at a dosage of from 50 mg to 300 mg/day, from 75 mg to 300mg/day, from 100 mg to 300 mg/day, from 150 mg to 250 mg/day, or at 150mg/day, 175 mg/day, 200 mg/day, 225 mg/day, or 250 mg/day. In otheraspects, when used in combination with a JAK inhibitor such asruxolitinib, Compound 1, or a pharmaceutically acceptable salt thereof,may be formulated at a dose of from 50 mg to 500 mg for e.g.,administration once, twice, or three times daily. For example, incombination therapies, Compound 1 may be administered at a dosage offrom 50 mg to 300 mg/day, from 75 mg to 300 mg/day, from 100 mg to 300mg/day, from 100 mg to 200 mg/day, or at 100 mg/day, 125 mg/day, 150mg/day, 175 mg/day, or 200 mg/day.

EXEMPLIFICATION

Compound 1 can be obtained following the procedures described in U.S.Pat. No. 8,796,261 and WO 2015/195862, both of which are incorporatedherein by reference.

Inhibitory Effect on Cytokine Release In Vitro

Compound 1 was assessed for its ability to suppress the expression ofNF-κB target genes in two experiments. In one experiment, THP-1 acuteleukemia cell lines were exposed to lipopolysaccharide treatment andthen Compound 1 for 16 hours. IL6 release from the THP-1 acute leukemiacells was inhibited, with an IC₅₀ of 0.069 μM. In the other experiment,the ability of Compound 1 to suppress both IL6 and IL10 expression inTMD8 ABC-DLBCL cells was investigated (data on file). TMD8 cells wereincubated with DMSO or 1.6 μM Compound 1 for 6 or 24 hours. RNA was thenextracted from the cells and quantified using qRT-PCR. As shown in FIG.1, Compound 1 substantially suppressed mRNA transcription of both IL6and IL10 after 6 and 24 hours of treatment.

Effect of Compound 1 as a Single Agent on Megakaryocyte Differentiation

The effects of Compound 1 on megakaryocyte differentiation andproliferation were evaluated using CD34+ cells isolated from healthydonor bone marrow (data on file). The CD34+ cells were grown inmegakaryocyte differentiation serum-free stem cell differentiation basemedium with a megakaryocyte-driving cytokine cocktail for 14 days withDMSO or Compound 1 at concentrations ranging from 3 nM to 500 nM. Thecells were then stained for CD34 (progenitor marker), CD45 (leukocytemarker) and CD41a (mature megakaryocyte marker) and assessed by FACS forviability and marker expression. CD41a expression and cell size wereused as markers of megakaryocyte differentiation. Compound 1 reduced thenumber of cells with high CD41a expression in a concentration-dependentmanner. The shift from high to low CD41a expression began atapproximately 50 nM, with pronounced effects observed at 200 to 500 nM,as shown in FIG. 2. The loss of CD41a-high-expressing cells suggestsimpaired megakaryocyte differentiation and loss of maturemegakaryocytes.

Effects of Compound 1 Alone and in Combination with Ruxolitinib onMegakaryocyte Differentiation and Proliferation

In a similar experiment, CD34+ cells that were isolated from the bonemarrow of two healthy donors were incubated for 10 days in megakaryocytedifferentiation media with DMSO; Compound 1 alone, at a concentration of30 to 500 nM; ruxolitinib alone at concentration of 8 to 1000 nM; orCompound 1 in combination with ruxolitinib at the same concentrationsthey were tested alone (data on file). The cells were then harvested forFACS analysis with live/dead stain and gating with CD34 (progenitormarker) and CD41a and CD42b (mature megakaryocyte markers). WhileCompound 1 showed limited effects on overall viability (percent of livecells by live/dead stain), it demonstrated potent effects on overallcell proliferation (total live count) and megakaryocyte differentiation(percent of cells double positive for CD41a and CD42b), which led to anoverall loss of live mature megakaryocytes (mean EC₅₀ of 28 nM; Table1).

In contrast to Compound 1, ruxolitinib exerted effects on megakaryocytedifferentiation at a similar concentration that killed the progenitorcells (mean EC₅₀ values of 526 and 644 nM, respectively; Table 1),suggesting the inhibitory effects of ruxolitinib on megakaryocytes arebased on its cytotoxicity. When serial dilutions of Compound 1 werecombined with serial dilutions of ruxolitinib, an additive inhibitoryeffect was observed on megakaryotcyte differentiation (FIG. 3). Asimilar additive effect was seen on overall cell proliferation where themean EC₅₀ for Compound 1 decreased from 38 to 17 nM (extrapolated),below the lowest dose tested in the presence of 250 nM ruxolitinib,indicating that concentrations of Compound 1 and ruxolitinib near theirIC₅₀ values for megakaryocyte differentiation and proliferation wereeffective at reducing the quantity of the other agent needed to elicitthe same effect.

TABLE 3 Compound 1 EC50 values following 10 days of treatment of CD34+cells Compound 1 EC₅₀ (nM) Ruxolitinib EC₅₀ (nM) Parameter Donor 1 Donor2 Mean Donor 1 Donor 2 Mean Viability 300 >500 400 676 611 644 Totallive count 43 32 38 288 259 274 Megakaryocyte 60 131 96 517 535 526differentiation Megakaryocyte 26 29 28 312 258 285 live count

Reduction in Cytokine Levels in Peripheral Blood

A panel of selected BET target genes (CCR1, CCR2, IL8, FN1, CSF1R andTHBS1) was evaluated in peripheral blood samples from patientsparticipating in the Compound 1 Phase 1 clinical studies, in order todetermine the relationship between systemic exposure of Compound 1 andsuppression of these BET inhibitor-sensitive genes. Gene expressionanalysis, along with the Compound 1 plasma concentration versus timedata, shows that there is a time- and concentration-dependentrelationship. Consistent with non-clinical data, Compound 1-inducedchanges in expression were most consistently observed for IL8 and CCR1at 2 hours post treatment, indicating the rapid effects of BETinhibition on transcription. Examples of the exposure-responserelationships for CCR1 and IL8 are presented in FIG. 4. The data shownincludes samples taken from patients with lymphoma who were treated withCompound 1 in Study 0610-01. Gene expression values were normalized tothose measured at a single time point pre-treatment (100%). This datademonstrated the rapid on-target effects of BET inhibition on keypro-inflammatory genes and supports the use of this clinical biomarkerassay.

Initial Human Clinical Data Set Clinical Signs Overview of Activity inPatients with Myelofibrosis

Four patients with myelofibrosis were enrolled in a human trial thatdemonstrated clinical benefit which extended at least 6 months. Twomyelofibrosis patients to enroll (Patients 245 and 246) receivedCompound 1 in combination with ruxolitinib and received 18 treatmentcycles (11 months of treatment). The other two patients to enroll(Patients 247 and 248) received 10 cycles of Compound 1 as monotherapy(6 months of treatment). All four patients experienced a reduction intheir constitutional symptoms along with a decrease in spleen volume andan increase in hemoglobin. One of the patients who was transfusiondependent at study entry became transfusion independent (defined as >12weeks without the need for a red blood cell (RBC) transfusion. Indeed, 7months had elapsed since their last transfusion.

All four patients experienced an increase in their hemoglobin levelswith multiple treatment cycles with Compound 1. In addition, one of thepatients who entered the study with uncontrolled thrombocytosis(baseline platelets were 895×10⁹/L) experienced a normalization of theirplatelet counts within the first month of monotherapy treatment withCompound 1. The patient's platelet counts remained normal for more than20 weeks. This patient's thrombocytosis was also accompanied by severeheadaches requiring multiple hospital admission. Following treatmentwith Compound 1, however, the patient's headaches were resolved. Briefnarratives for all four patients treated for at least 6 months arepresented below.

Combination Therapy Arm

Patient 245, a 66 year-old female was diagnosed with myelofibrosis inMay 2014, remained treatment naïve until January 2016 when she initiatedtreatment with ruxolitinib 15 mg twice daily (BID). Panobinostat wasadded in February 2016 and discontinued in March 2017, due to thedevelopment of anemia. From March 2017, while on ruxolitinib alone, thepatient became resistant to ruxolitinib, with her spleen increasing 25%in size.

At entry into the study with Compound 1, her spleen volume by MRI was1404 cc and was 12 cm by palpation. The patient presented with earlysatiety, night sweats, and dyspnea at the start of the study. Within 4months of treatment with Compound 1, 125 mg QD and ruxolitinib 15 mgBID, the patient had resolution of early satiety; her spleen was 5 cm bypalpation and her liver was no longer palpable. The lowest spleen volumeby MRI was 1144 cc, a 19% reduction, at the 6-month MM. The dose ofruxolitinib was reduced on Cycle 10 to 7.5 mg BID to address decreasingplatelet count. Her platelets counts gradually improved following thedose reduction of ruxolitinib and remained below the protocol-specifiedcriteria of 100×10⁹/L for two treatment cycles to permit a dose increasein Compound 1.

Patient 246 is a 53 year-old female who was diagnosed with myelofibrosisin 2009. During 2002 and 2006, the patient cycled between epoetin alfa,lenalidomide and thalidomide then received lenalidomide for 7 yearsuntil 2013. She required RBC transfusions during 2013 and initiatedinterferon in 2014, which allowed her to become transfusion independent.Interferon was discontinued almost a year later due to fatigue. Thepatient remained transfusion independent and without further treatmentuntil late 2016 when they once again became transfusion dependent.Ruxolitinib 5 mg BID was started in January 2017. Ruxolitinib wasincreased to 10 mg BID in April 2017, but the patient remainedtransfusion dependent and symptomatic. She was considered ruxolitinibresistant due an increasing spleen size and exacerbated symptoms(extreme fatigue, shortness of breath, distress on exertion, occasionalnausea and night sweats) while on ruxolitinib therapy.

When Patient 246 initiated combination treatment with Compound 1 125 mgQD and ruxolitinib 10 mg BID her spleen volume was 607 cc by MRI and 2cm by palpation and she required regular transfusions (2 units of RBCevery 3-4 weeks). The dose of Compound 1 was titrated up to 175 mg QDafter five treatment cycles and within 7 months of combination therapy,the patient had become transfusion independent (defined as >12 weekswithout a transfusion and hemoglobin >8 g/dL; see FIG. 5), which hasbeen maintained for >30 weeks (most recent hemoglobin measurement was10.9 g/dL). She has also experienced a clinically meaningful improvementin her associated constitutional symptoms (fatigue and dyspnea) and hashad an incremental decrease in spleen volume, achieving a 37% reductionin spleen volume by Cycle 12 (380 cc).

Monotherapy Arm

Patient 247 is a 46 year-old female who was diagnosed with myelofibrosisin April 2014. In 2009, it was suspected that the patient had essentialthrombocytosis (ET) for which she received hydroxyurea treatment fromApril 2009 to December 2017. The patient also received one month ofepoetin alpha in 2015, three months of imetelstat in 2016 and fourmonths of pembrolizumab in 2017. Ruxolitinib was administered fromOctober 2015 to May 2016. Ruxolitinib was discontinued due to worseningsymptomatic splenomegaly, anemia, leukocytosis, and thrombocytosis.

Upon entry into the study with Compound 1, Patient 247 had a spleenvolume of 858 cc by Mill and 5 cm by palpation and a host ofconstitutional symptoms, including: abdominal discomfort, dyspnea onexertion, early satiety, fatigue, headaches, night sweats, dizziness,insomnia, pruritus, and bone pain. The patient also presented withuncontrolled thrombocytosis at study entry (platelet count of 895×10⁹/Lat baseline) despite hydroxyurea. In addition, the patient hadexperienced persistent and debilitating headaches that required multiplehospital admissions for pain control. Their platelets were normalizedafter receiving their first two weeks of Compound 1 monotherapy(183×10⁹/L) and have remained within the normal range for the remainderof the time they have been on study (see FIG. 6). Within 2 months onCompound 1 monotherapy, the patient's severe headaches had resolved;their night sweats were less frequent; and a 37% reduction in symptomswas assessed by the Myeloproliferative Neoplasm Symptom (MNS) score.Their ECOG performance score decreased from 2 to 1 after two treatmentcycles with CPI-0610 and a 25% decrease in spleen volume (640 cc) wasassessed by MRI after 8 treatment cycles, the most recent measurement.

Patient 248, a 76 year-old male who was diagnosed with myelofibrosis inSeptember 2011 was treated with fresolemunib (December 2011 to October2012) and itacitinib (December 2012 to July 2014). Ruxolitinib 5 mg BIDwas initiated in January 2015 and increased to 15 mg BID in December2015. Ruxolitinib was discontinued in September 2016 because the patientwas experiencing generally worsening fatigue, anemia andthrombocytopenia. Subsequent to ruxolitinib treatment, the patientreceived imetalstat from June 2016 through March 2017, followed bypembrolizumab from June to October 2017.

Upon entry into the study with Compound 1, the patient had a spleenvolume of 1148 cc by MRI and 5 cm by palpation. Their constitutionalsymptoms at study entry included fatigue, early satiety, and difficultyconcentrating. The patient did not tolerate the 225 mg QD starting doseof Compound 1 (he experienced nausea, diarrhea, malaise and dizziness),requiring dose interruption after the first 5 doses of Cycle 1. Thepatient was reinitiated with a reduced Compound 1 dose of 175 mg at thebeginning of Cycle 2, which has been tolerated for the remaining time onstudy (>6 months). While the patient's spleen has shown minimal changethrough palpation a spleen volume reduction of 11% (1023 cc) wasmeasured by MRI after 3 months on treatment with Compound 1. Their MNSscore improved 19% after 2 months on the 175 mg QD dose and after 6months of Compound 1 treatment his bone marrow fibrosis grade decreasedfrom MF-2 at baseline to MF-1 based on a local pathologist's assessment.

Human Phase 2 Clinical Data Set

A Phase 2 study of Compound 1 in subjects with myelofibrosis (MF) wasconducted. Three treatment arms were studied:

Arm 1=monotherapy with Compound 1 in patients (pts) who are no longer onRux and are with refractory, intolerant, or ineligible. In this armpatients were further stratified based on transfusion dependence status[transfusion dependent (TD), defined as an average of ≥2 units per monthover 12 wks, or non-TD cohorts]. Primary endpoint: spleen volumeresponse (SVR) for non-TD cohorts or TD to transfusion independence (TI,no transfusion for consecutive 12 wks) conversion for TD cohorts;secondary endpoints: change in total symptom score (TSS) per MFSAF v4.0,patient global impression of change (PGIC), safety and PK; additionalendpoints: changes in proinflammatory Ck levels, BM morphology andmutant allele burden.

Arm 2=combination add-on treatment of Compound 1 in patients (pts) whoare already being administered Rux. In this arm patients were furtherstratified based on transfusion dependence status [transfusion dependent(TD), defined as an average of ≥2 units per month over 12 wks, or non-TDcohorts]. The starting dose of Compound 1 was 125 mg daily on days 1-14of a 21-day cycle in both arms. Primary endpoint: spleen volume response(SVR) for non-TD cohorts or TD to transfusion independence (TI, notransfusion for consecutive 12 wks) conversion for TD cohorts; secondaryendpoints: change in total symptom score (TSS) per MFSAF v4.0, patientglobal impression of change (PGIC), safety and PK; additional endpoints:changes in proinflammatory Ck levels, BM morphology and mutant alleleburden.

Arm 3=combination treatment with Compound 1 and Rux in patients (pts)who have not previously been administered a JAK inhibitor, i.e., JAKinhibitor treatment naïve. In this arm, key eligibility criteria ofincluded JAKi naïve myelofibrosis (MF) patients (pts) with DynamicInternational Prognostic Scoring (DIPSS) score int-1 or higher, ECOGperformance status ≤2, platelet counts (PLT) ≥100×109/L, peripheralblood blast count <10%, anemia (hemoglobin <10 g/dL), ≥5 cm palpablespleen, ≥2 symptoms measurable (score ≥3) or a total symptom score (cusing the MFSAF v4.0. Primary endpoint: spleen volume response (SVR);key secondary endpoints: change in TSS, safety and PK; additionalendpoints: changes in proinflammatory cytokine (Ck) levels, BMmorphology and mutant allele burden.

The starting dose of Compound 1 was 125 mg given orally, once daily for2 weeks on/1 week off in a 21-day dosing cycle. Two separate time pointsfor data analysis occurred for at least Arms 1 and 2 and are discussedbelow: one at approximately 12 weeks of treatment (first assessment ofspleen volume by Mill or CT and every 12 weeks thereafter) and anotherat approximately 24 weeks of treatment (first assessment of bone marrowbiopsy and every 24 weeks thereafter). Patients were added to the studyfollowing the first time point. An overview of the study profile isshown in FIG. 7.

Time Point 1:

Arm 1 and Arm 2—Compound 1 as Monotherapy or Add-On to Ruxolitinib inPatients with Refractory or Intolerant Advanced Myelofibrosis

Demographics and results are as follows: At baseline, median age: 69years (41-88), gender: 28 (58%) male, ECOG ≤1: 45 (94%) patients,primary MF: 33 (69%) patients, DIPSS score high: 10 (21%) patients,median platelet: 199×109/L (77-895), 34 (71%) patients with Hgb <10g/dL, median spleen volume: 2183 cc (123-3909), median TSS: 17.6(1.4-56), 46 (96%) patients had ≥1 JAK2/MPL/CALR mutations, and 34 (71%)patients had HMR (high molecular risk) mutations. 33 (69%) patients ontreatment for ≥12 wks, 4 on treatment for >18 months.

Spleen volume reduction observed in 29 of 31 (94%) patients (median bestchange: −17% [range: −50.7, 10.2]) (FIG. 8, panel A). TSS improvementwas reported in 26 of 28 (93%) patients (median best change: −46.4%[range: −95.3%, 27%]), 11 (39%) patients with ≥50% TSS improvement (FIG.8, panel B). PGIC improvement score in 28 of 33 (85%) patients; 21 (64%)reported much or very much improved scores. Increase in hemoglobin by1.5 mg/dL post-baseline observed with both Compound 1 monotherapy (4 of8, 50%) and Compound 1 with ruxolitinib (4 of 25, 16%)). Improvement inBM fibrosis and/or reticulin by ≥1 Gr reported in 7 of 12 (58%)evaluable patients with baseline and 1 post-baseline biopsy and as earlyas 6-months of Compound 1 treatment. 4 TD patients in Arm 2 treated withCompound 1 with ruxolitinib converted to TI-2 of whom are TI for >36wks, no longer anemic, and showed spleen volume reduction, improvementin symptom and BM fibrosis; 12 additional patients are being monitoredfor potential TI conversion. 41 patients remain active on treatment and7 patients discontinued, including 1 patient, initially transplantineligible, underwent stem cell transplantation after 6 cycles ofCompound 1 with ruxolitinib treatment. Most common (≥20%)treatment-emergent adverse events (TEAE) of any Gr include diarrhea,nausea, cough and upper respiratory tract infection. Most common (≥5%)≥3 Gr TEAE include anemia (8.3%) and thrombocytopenia (8.3%,asymptomatic, non-cumulative and generally reversible).

This data indicates that Compound 1 alone or “add-on” to ruxolitinib isgenerally well-tolerated and provides clinical benefits in MF patientswith inadequate responses or who are refractory to ruxolitinib.Improvement in BM fibrosis and anemia responses indicate the potentialfor meaningful disease modification.

Arm 3—Compound 1 and Ruxolitinib in JAK Inhibitor Treatment NaïveMyelofibrosis Patients

Demographics and results are as follows: Baseline median age: 71 years(52-76), gender: 8 male (72.7%), ECOG ≤1: 10 (90.9%) patients, primaryMF: 8 (72.7%) patients, DIPSS score: int-1/int-2/high: 2/7/2 patients,median platelet: 368×109/L (112-951), 9 (81.8%) patients with hemoglobin<10 g/dL, median spleen volume: 1379 cc (580-2807), median TSS: 11.8(4.1-17), driver mutations: 11 (100%) with ≥1 JAK2/MPL/CALR mutations,HMR (high molecular risk) mutations: 6 (56%) patients, and ≥3 mutations:4 (36%) patients. All 4 (100%) patients on treatment for ≥12 weeksachieved ≥35% spleen volume reduction (median: −52.4%, [range −68.7%,−42.7%]) and all 4 patients (100%) achieved ≥50% improvement in TSS(median best change: −79.35% [range −90.2%, −70.1%]). See FIG. 9,where * is data post cut off-date. A reduction of proinflammatory Ck,including IL-18 and CRP, was also observed. Safety data from the first 6patients who received treatment for at least 1 cycle were reviewed: noDLTs or grade ≥3 thrombocytopenia was observed. The most commontreatment-emergent adverse events (TEAE) observed in ≥2 patients includeanemia (1 grade 3), fatigue (all ≤grade 2), and non-cumulativereversible thrombocytopenia (all ≤grade 2).

Overall, the combination of Compound 1 and ruxolitinib was generallywell-tolerated demonstrating that the safety of this combination isacceptable in JAKi naïve MF patients with anemia. Early clinicalactivity was observed with the combination: all 4 evaluable patientsachieved both ≥35% SVR and ≥50% improvement in TSS as early as 3 monthsafter treatment. Available data in JAKi naïve anemic MF patients, apopulation with poor prognosis, along-with additional information onreduction in pro-inflammatory Ck and BM fibrosis improvement in Compound1 treated patients in ruxolitinib refractory MF, collectively indicatethat addition of Compound 1 to ruxolitinib could have disease-modifyingeffects in JAKi naïve MF patients.

An expanded patient population (n=15) for Arm 3 were treated for atleast 12 weeks. About 80% of patients achieved ≥35% SVR with an averageof about −49.7%. See FIG. 16. Hgb was less than 10 g/dL and DIPSS scorewas int-2 or higher. In addition, about 71% of patients had a TSS of ≥10(see FIG. 17) and about 80% of patients had an overall improvement inPGIC (see FIG. 18).

Time Point 2

Patients were added to the study following the first data cut andtreatment was continued for approximately 25.9 weeks (median, range:0.4, 116.6). A summary of the results from the trial are provided below.

FIGS. 10A-C illustrates the 24 week results from treatment with Compound1 as an add-on to ruxolitinib (Arm 2) in patient's with refractory orintolerant myelofibrosis subjects who were transfusion dependent at thestart of therapy. At 24 weeks, about 35% of patients converted fromtransfusion dependent to transfusion independent as represented by theupward arrows (FIG. 10A). The average spleen volume reduction was about−24.9%. About 76.5% of patients had improvement in disease symptoms pertotal symptom score (TSS) about 75% of patients had improved PatientGlobal Impression of Change (PGIC). See FIGS. 10B and 10C. Further,about 63% of patients showed improvement in bone marrow fibrosis (datanot shown).

FIGS. 11A-D shows the absolute hemoglobin values and transfusionrequirements for representative patients from Arm 2. The average time ofconversion from transfusion dependent (TD) to transfusion independent(TI) was about 14 weeks. The average transfusion free timepost-conversion was about 14 weeks with a maximum of 85 weeks. About 41%of patients had greater than or equal to a 50% reduction in transfusionintensity.

FIGS. 12A-C illustrates the 24 week results from treatment with Compound1 as an add-on to ruxolitinib (Arm 2) in patients with refractory orintolerant myelofibrosis subjects who were non-transfusion dependent atthe start of therapy. At 24 weeks, the average spleen volume reductionwas about −10.9% (FIG. 12A). About 38% of patients had improvement indisease symptoms per total symptom score (TSS) about 69% of patients hadimproved Patient Global Impression of Change (PGIC). See FIGS. 12B and12C. Further, about 25% of patients showed improvement in bone marrowfibrosis (data not shown).

FIGS. 13A-C illustrates the 24 week results from treatment with Compound1 monotherapy (Arm 1) in patients with refractory or intolerantmyelofibrosis subjects who were transfusion (Cohort 1A) ornon-transfusion dependent (Cohort 1B) at the start of therapy. At 24weeks, the average spleen volume reduction was about −3.2% for TDpatients and about −26% for non-TD patients (FIG. 13A). About 60% ofnon-TD patients had improvement in disease symptoms per total symptomscore (TSS) about 50% of TD and 100% of non-TD patients had improvedPatient Global Impression of Change (PGIC). See FIGS. 13B and 13C.Further, about 25% of TD patients showed improvement in bone marrowfibrosis (data not shown).

The improvement in bone marrow fibrosis from Arms 1 and 2 were alsoevaluated. See FIG. 14. It was found that about 38% of subjects had bonemarrow fibrosis improvement with about 32% having improvement as earlyas 6-months. Additionally, the best improvement in bone marrow fibrosiswas seen in Cohort 2B (See FIG. 7) at 63% of patients havingimprovement, i.e., those patients who were transfusion dependent at thestart of therapy and were given Compound 1 as an add-on to ruxolitinib.

Pro-inflammatory cytokine levels showed a trend toward normalizationwithin 14 days for both monotherapy and combination therapy arms. SeeFIGS. 15A-F.

While have described a number of embodiments of this, it is apparentthat our basic examples may be altered to provide other embodiments thatutilize the compounds and methods of this disclosure. Therefore, it willbe appreciated that the scope of this disclosure is to be defined by theappended claims rather than by the specific embodiments that have beenrepresented by way of example.

The contents of all references (including literature references, issuedpatents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated herein in their entireties by reference. Unless otherwisedefined, all technical and scientific terms used herein are accorded themeaning commonly known to one with ordinary skill in the art.

The invention claimed is:
 1. A method of treating myelofibrosis in asubject comprising administering to the subject a therapeuticallyeffective amount of2-((4S)-6-(4-chlorophenyl)-1-methyl-4H-benzo[c]isoxazolo[4,5-e]azepin-4-yl)acetamide,or a pharmaceutically acceptable salt thereof.
 2. The method of claim 1,wherein the subject has previously undergone treatment with a januskinase (JAK) inhibitor.
 3. The method of claim 1, wherein the subject isprogressed/relapsed to a JAK inhibitor.
 4. The method of claim 1,wherein the subject is refractory/resistant to a JAK inhibitor.
 5. Themethod of claim 1, wherein the subject is intolerant to a JAK inhibitor.6. The method of claim 1, wherein the subject has previously undergonetreatment with ruxolitinib.
 7. The method of claim 1, wherein thesubject is a janus kinase (JAK) inhibitor naïve subject.
 8. The methodof claim 1, further comprising administering to the subject atherapeutically effective amount of a janus kinase (JAK) inhibitor. 9.The method of claim 8, wherein the subject is a janus kinase (JAK)inhibitor naïve subject prior to treatment.
 10. The method of claim 8,wherein the JAK inhibitor is ruxolitinib.
 11. The method of claim 1,wherein the subject is cytopenic.
 12. The method of claim 1, wherein thesubject is anemic.
 13. The method of claim 1, wherein the subject has ahemoglobin count of less than 10 g/dL.
 14. The method of claim 1,wherein the subject is thrombocytopenic.
 15. The method of claim 1,wherein the subject's platelet count is less than 120,000 platelets/μL.16. The method of claim 1, wherein the subject is thrombocytemic. 17.The method of claim 1, wherein the subject's platelet count is more than400,000 platelets/μL.
 18. The method of claim 1, wherein the subject'splatelet count is more than 500,000 platelets/μL.
 19. The method ofclaim 1, wherein the subject is neutropenic.
 20. The method of claim 1,wherein the subject's absolute neutrophil count is less than 1000neutrophils/μL of blood.
 21. The method of claim 1, wherein the subjecthas an enlarged spleen or liver.
 22. The method of claim 1, wherein thesubject is suffering from abdominal discomfort, dyspnea on exertion,early satiety, fatigue, headaches, night sweats, dizziness, insomnia,pruritus, or bone pain.
 23. The method of claim 1, wherein the subjectis transfusion dependent.
 24. The method of claim 1, wherein the subjectis administered from 100 mg/day to 300 mg/day of2-((4S)-6-(4-chlorophenyl)-1-methyl-4H-benzo[c]isoxazolo[4,5-e]azepin-4-yl)acetamide.
 25. The method of claim 1,wherein the subject is administered2-((4S)-6-(4-chlorophenyl)-1-methyl-4H-benzo[c]isoxazolo[4,5-e]azepin-4-yl)acetamideonce per day.
 26. The method of claim 1, wherein the subject isadministered a pharmaceutically acceptable salt of6-(4-chlorophenyl)-1-methyl-4H-benzo[c]isoxazolo[4,5-e]azepin-4-yl)acetamide.27. The method of claim 1, wherein the subject is administered2-((4S)-6-(4-chlorophenyl)-1-methyl-4H-benzo[c]isoxazolo[4,5-e]azepin-4-yl)acetamide.28. The method of claim 1, wherein the subject's platelet count is morethan 450,000 platelets/μL.
 29. The method of claim 1, wherein thesubject's platelet count is more than 600,000 platelets/μL.
 30. Themethod of claim 1, wherein the subject is administered from 50 mg/day to500 mg/day2-((4S)-6-(4-chlorophenyl)-1-methyl-4H-benzo[c]isoxazolo[4,5-e]azepin-4-yl)acetamideor a pharmaceutically acceptable salt thereof.